Thiolated polypeptide compound derived from a tetanus toxin fragment, the process for its obtention and its applications

ABSTRACT

A new thiolated polypeptide compound derived from a fragment of tetanus toxin, the process for its obtention and its applications. This compound consists of the IIc fragment of tetanus toxin, having at least one -SH group either directly or indirectly bound thereto. It is usable as a specific neuropharmacological transport agent for transporting a medicine to the central nervous system, as a specific labelling agent for neuronal cells or for diagnosis purposes. It can be coupled with a medicine or a labelling agent.

The present invention relates to a new thiolated polypeptide compoundderived from a tetanus toxin fragment, to the process for its obtentionand its applications.

With regard to the tetanus toxin itself or its anatoxin, it was alreadyproposed to use them notably for obtaining vaccines or assay reagents.In order to illustrate this prior art following references will becited:

FR patent for addition No. 74,16,936 published under No. 2,270,891,which relates to a process for obtaining vaccines by treatment of atoxic product with glutaraldehyde. This process consists in treating atoxic product with glutaraldehyde in effecting the polymerisation of alimited number of molecules of said product and the detoxification ofsaid product. In this process tetanus toxin may be used as toxicproduct.

FR patent application No. 77,29,186, published under No. 2,366,569,relates to an immunochemical process for assaying haptens, wherein areused a particle sensitized by an antibody, prepared by sensitizing fineparticles by an antibody of the hapten to be assayed and ahapten-carrier conjugate. The carrier of this conjugate may be notablytetanus toxoid.

This hapten-carrier conjugate is used as reagent in an immunochemicalprocess and also for immunization of an animal in order to obtaincorresponding antibodies [see on page 4, lines 20 to 32].

The tetanus toxoid is therefore used as a carrier of the hapten in thebody of the animal for obtaining antibodies. However, there exists noteaching in this FR patent about a particular fragment of tetanus toxinand its possible use as axonal transport agent for drugs.

GB patent No. 2,013,690 relates to an antigen for early pregnancy testand contraceptive vaccine. This antigen is obtained from the β-subunitof human chorionic gonadotrophin by reducing and cleaving of three,four, five or six of intrachain disulphide bounds of said β-subunit,alkylating of the thus reducing intrachain disulphide groups andisolating of the produced antigen. This antigen may be coupled with aprotein or a hapten to enhance its immunological specificity. Tetanustoxoid is cited as suitable protein.

GB patent No. 1,492,445 relates to a composition comprising a conjugateof a subject-compatible immunogenic carrier and an immunochemicallypurified hormone derivative. The tetanus toxoid is used as carrier inthis composition.

DE-OS patent application No. 1,617,880 relates to a process forobtaining bioactive organotrope substances, particularly drugs. Thisprocess consists in making a conjugate of a biologically activesubstance with organotrope receptive substances obtained from cellularmembranes or antibodies. The toxins may be used as organotropesubstances.

Furthermore, it was proposed to use thiolated proteins as drug carriers.Reference may be made to U.S. Pat. No. 3,171,831 which relates tothiolation of proteins by reaction with homocysteine thiolactone in thepresence of a tertiary amine. The thiolated proteins thus obtained, forexample gelatin, may be used as carriers for drugs. According to example18 of this U.S. Pat. No. 3,171,831 the gelatin thus treated is used forencapsulating a pharmaceutical product which is sensitive to the acidenvironment of the stomach. The pharmaceutical product is therefore inthis case not coupled with the thiolated protein but coated with it.

On the other hand, it was disclosed in FR patent application No.76,37,367 published under No. 2,334,954 a reagent for immunoenzymaticdetermination. This reagent is composed by an antigen and an enzymecoupled by means of an ester of maleimidobenzoic acid andn-hydroxy-succinimide.

It is known that tetanus toxin is retrogradely transported to thecentral nervous system and the peripheral nervous system. In thisrespect, reference may be made to the article of BIZZINI et al entitled:"An antigenic polypeptide fragment isolated from tetanus toxin:chemicalcharacterization, binding to gangliosides and retrograde axonaltransport in various neuron sytems", which appeared in the "Journal ofNeurochemistry", 1977, vol. 28, pp 529-542, and to all the bibliographicreferences cited in this article.

Various studies have shown that tetanus toxin may be degraded or cleavedinto several fractions or subunits. For example, COHEN et al [TheJournal of Immunology vol. 104, No. 6 June 1970] have shown that thefreezing-thawing of the crude filtrate of Clostridium tetani cultureresults in a degradation of the molecule of tetanus toxin; the resultingdegraded tetanus toxin is practically devoid of toxicity and has aflocculating power lower than the one of tetanus toxin.

BIZZINI and RAYNAUD have also studied the subunits A-I, A-II, A-III andB-I, B-II and B-III of tetanus toxin. [C.R. Acd. Sc. Paris, T.279,1974series D, pp. 1809-1811 and Annales of Pasteur Institute Paris 126,159-176 (1975)]. French patent No. 74,36,622 (publication No. 2,249,679)discloses an immunogenic atoxic product obtained from tetanus toxin.This atoxic product is obtained by the treatment of tetanus toxin with aproteinase.

MORRIS et al. in the Journal of Biological Chemistry vol. 255 No. 13July 10, 1980 pp. 6071-6076 disclose the interaction of fragments B andC of Tetanus toxin with neural and thyroid membranes and withgangliosides. It is notably noted in this article that fragment C,obtained by papain digestion at 55° C., undergoes retrograde transportin a manner similar to that of intact tetanus toxin, whereas fragment Bdoes not similarly accumulate in the first distal segment to theligation but does appear to be taken up or internalized in a diffusepatern (see on page 6075, left column).

The principal physico-chemical properties of the different subproductsof tetanus toxin are given on page 230 of article entitled "Tetanustoxin" of BIZZINI in Microbiological Reviews, June 1979, p. 224-240 andnotably those of fragment C and fragment II_(c) which is obtained, bypapain digestion at a temperature at which no formation of fragment Coccurs.

BIZZINI et al have also isolated from frozen crude toxin a polypeptidefragment of the toxin which is identical, from the immunological pointof view, to the above mentioned fragments A-II and B-II, but differstherefrom by its size and toxicity [see in this respect Journal ofNeurochemistry, 1977, vol. 28, pp. 529-542]. This fragment, namedB-II_(b), is capable of binding to the gangliosides and to the synapticmembranes with an affinity which is even greater than that of tetanustoxin.

French patent application No. 79,29,289, discloses a new thiolatedpeptide compound derived from tetanus toxin, the process for itsobtention, and its applications. This thiolated polypeptide compoundconsists of the B-II_(b) fragment of tetanus toxin having bound theretoat least one --SH group; this compound substantially exhibits the sameproperties of axonal retrograde transport and of binding to the tetanustoxin receptors as the B-II_(b) fragment itself. This thiolatedpolypeptide compound is suitable as a neuropharmacological transportagent for conducting to the central nervous system pharmacological orchemotherapeutic agents. This polypeptide agent is also suitable as alabelling agent for neuronal cells. The properties of this thiolatedpolypeptide compound are also described in Brain Research 193 (1980)221-227.

There has been now found a new thiolated polypeptide compound derivedfrom another tetanus toxin fragment, which may be also used as aneuropharmacological transport agent to transport pharmacological orchemotherapeutic agents to the central nervous system.

The present invention therefore relates to a new thiolated polypeptidecompound, its obtention and its applications.

The thiolated polypeptide compound according to the invention is theII_(c) fragment of tetanus toxin having bound thereto at least one --SHgroup and substantially exhibiting the same properties of axonalretrograde transport and of binding to the tetanus toxin receptors asthe II_(c) fragment itself.

The B-II_(b) fragment of tetanus toxin is derived from a frozen filtrateof a culture of Clostridium tetani (Harvard strain No. 6037 ofCollection Nationale de Cultures de Microorganismes de l'InstitutPasteur in PARIS, FRANCE) by ultrafiltration for removing the substanceswith a molecular weight lower than 10,000, then by fractionating bymeans of ammonium sulfate and gel filtration.

A number of fragments similar to the B-II_(b) fragment may be obtainedby papain digestion of the whole tetanus toxin or of the heavy chainthereof (beta fragment). In this connection, reference may be made tothe following articles:

BIZZINI, B. and RAYNAUD, M. entitled "Immunological and biological studyof subunits of tetanus toxin" C.R. Acad. Sci. (Paris), 279, series D(1974) 1809-1812.

BIZZINI, B., International Symposium or Venoms and Toxins, March 4-6,1977, Bombay (India); abstract published in Toxicon (1978), volume 15,p. 141.

BIZZINI B., Tetaus toxin Microbiol. Rev. 43 (1979) 224-240.

The II_(c) fragment is obtained by digestion of the purified tetanustoxin by means of papain insolubilized on the gel, "Sepharose 4B",activated with cyanogen bromide according to the procedure disclosed inthe previously mentioned Compte Rendu de l'Acad/e/ mie des Sciences[T.279 December 1974; series D-1809], except that digestion is effectedonly for 4 hours.

Statement will be given hereinafter of the procedure to obtain theII_(c) fragment used as a starting compound for providing thepolypeptide compound according to the invention. There was used thepurified tetanus toxin derived from a filtrate of a 5-day culture ofClostridium tetani obtained by the process disclosed by:

BIZZINI, B. TURPIN A., and RAYNAUD, M. in the article entitledProduction and purification of the tetanus toxin Ann. Institut Pasteur,Paris 116 (1969) 686-712.

The purified tetanus toxin thus obtained assayed 3150 L_(f) (Limesfloculationis or Floculation unit) per mg of N and 1,2.10⁸ MLD (Minimumlethal dose) per mg of N.

120 mg of the purified tetanus toxin were treated for 4 hours at 37° C.in a water bath, with 200 units of inslublized papain, under occasionalstirring.

At the end of the digestion period, the papain was immediately removedby centrifugation for 5 minutes at 10,000×g in a centrifugal machinerefrigerated to 4° C.

The supernatent was filtrated on a column of "Sephadex G-100" (2.5×80cm) buffered with 0.1 M tris, HCl buffer, pH 8, containing 1 M of NaCl.The effluent was continuously controlled at 280 nm and collected in 2ml-fractions. The fractions corresponding to the peak, having amolecular weight close to 45,000, were combined together. The volume ofthe combined fractions was reduced to 2 ml before filtration on a column(2.5×80 cm) of polyacrylamide-agarose gel known under the trade name"Ultrogel AcA 54", using the same buffer. The fractions representing themain peak were combined together and the resulting volume was reduced to3 ml by dialysis against a solution containing 50% of polyethyleneglycol 20,000 (sigma).

The undigested toxin or the I_(bc) fragment liable to contaminate theII_(c) fragment were adsorbed on an affinity column of "Sepharose" gelactivated with cyanogen bromide which previously had coupled thereto,covalently, the IgG fraction of an anti-I_(bc) serum. The unadsorbedfraction present in the filtrate constitutes the II_(c) fragment. Thisfragment is not toxic to mice at a dose of 1 mg at one time.

The affinity column was prepared according to the following procedure.The IgG fraction was isolated by precipitating the anti-I_(bc) fragmentantiserum. The anti-fragment I_(g) G fraction assayed 1,500 L_(f) perml. The IgG fraction was coupld to "Sepharose 4B" activated withcyanogen bromide according to the procedure disclosed by Wilchek et al."A general method for the specific isolation of peptides containingmodified residues, using insoluble antibody columns. Biochemistry 10(1971) 2828-2834."

The II_(c) fragment has the following physicochemical properties:

molecular weight of about 46,000 it

interacts with gangliosides

it is free of toxicity

it interacts with synaptic membranes isolated from rat spinal cord.

The immunological properties of the II_(c) fragment are as follows:

the II_(c) fragment rise to a cross reaction with the tetanus toxin andthe heavy chain (β fragment) thereof, The II_(c) fragment gives rise toan identity reaction with the B-II_(b) fragment; the II_(c) fragmentgives rises to a non-identity reaction with the I_(bc) fragment and thelight chain (α fragment).

The main characteristics of the II_(c) fragment are summarized in thearticles by B. BIZZINI (Microbiological Reviews, June 1979, pp. 224-240and Toxicon (1978), Vol. 15, p. 141) wherein it is stated that thefragments similar to the B-II_(b) fragment may be obtained by digestionby means of papain and that those fragments probably contain the portionof the heavy chain which is involved in the binding of the toxinmolecule to its receptors in the nervous cell. On the other hand, it isstated that the tetanus toxin fragments, which still have the ability tobind with gangliosides should undergo axonal retrograde transport as thewhole toxin does.

It was now found that the II_(c) fragment may be bound to synapticmembranes and could undergo axonal retrograde transport to the centralnervous system. The thiolated II_(c) fragment, which is the object ofthe present invention, will also undergo axonal retrograde transport tothe central nervous system and will bind to the synaptic membranes.Since in this field any chemical modification might inhibit thepharmacological properties of a fragment, it was not at all obvious,even in view of the teachings relating to the B-II_(b) fragment, thatthe thiolated polypeptide compound according to the invention could alsobe used as an axonal retrograde carrier for pharmacological orchemotherapeutic agents or as a diagnosis reagent, either alone or asassociated with another substance adapted to evidence, for example, aspecific antigen in the central nervous system.

The present invention therefore relates to a new thiolated polypeptidecompound consisting of the II_(c) fragment bearing at least one --SHgroup, suitable especially as a neuropharmacological transport agent andas a specific labelling agent for neuronal cells.

Similarly to the process for obtaining the thiolated polypeptidecompound according to FR patent No. 79,29,289, the --SH group or groupsis or are directly or indirectly bound to the II_(c) fragment. Ingeneral, taking into account the process for its obtention, whichinvolves a thiolation, the binding of the --SH groups will occur throughthe residue of the thiolation agent. Besides, the latter is bound to theII_(c) fragment through the --NH₂ groups carried thereby.

The thiolated polypeptide compound according to the present invention isproduced by thiolation of the II_(c) fragment obtained by the abovedescribed process.

The thiolation of the II_(c) fragment can be carried out by conventionalmeans permitting the introduction of --SH groups on a moleculecomprising amino groups, but for the purposes of the invention, themeans in question should not denature the properties of axonal transportand of binding to the specific receptors of the tetanus toxin in thecentral nervous system, of the II_(c) fragment.

By way of example, it will be mentioned that the thiolation of theII_(c) fragment can be achieved with the following thiolation agents:

4-methyl-mercaptobutyrimidate: ##STR1## (Biochemistry vol. 17 No. 8,1978) 2-iminothiolane (Schramm H. J. and D/o/ lffer T. (1977)

Z. Physiol. Chem. 358; 137-139).

N-acetylhomocysteine thiolactone (AHT) (see J. Am. Chem. Soc. 1960, 82,565-571) ##STR2## S-acetyl-mercaptosuccinic anhydride (AMS) (J.Am.Chem.Soc.1959,81, 3802-3803) ##STR3##

On the other hand, it will be mentioned that the known processes ofthiolation consisting of a dithiopyridylation step and a reduction stepare unsuitable for the purposes of the invention. Indeed, the propertiesof axonal transport and of binding of the thus thiolated II_(c) fragmentare modified in the course of the reduction step.

For example, the thiolation effected by reaction with theN-succinimidyl-3-(2-pyridyl-dithio)-propionate and by reduction of thedithiopyridylated compound so obtained, for example according to theprocedure described by CARLSSON et al [Bioch, J. (1978) 173 723-724] isunsuitable for the purposes of the invention.

In order to be more precise, it will be stated that the thiolatedpolypeptide compound according to the invention comprises one or moreZ--SH groups, in which Z is the residue of the thiolation agent.

Thus, if one of the thiolation agents mentioned above is employed, Zthen represents: ##STR4##

The thiolation of the II_(c) fragment is achieved on the NH₂ groupsthereof.

It has been found that the thiolated polypeptide compound according tothe invention is suitable as a neuropharmacological transport agent fortransporting pharmacological or chemotherapeutic agents to the centralnervous system.

In order to allow the transport of a medicine to the central nervoussystem by means of the agent according to the invention, this medicinemust be bound to the thiolated polypeptide compound, employed as atransport agent, without of course modifiying the pharmacologicalproperty of the medicine or the property of the II_(c) fragment to bebound to the specific receptors of the tetanus toxin in the centralnervous system. The term "Medicines" is intended to designate, accordingto the invention, any substances having pharmacological properties, suchas pharmacological agents, chemotherapeutic agents and the like. Themedicines which may be bound according to the invention to thepolypeptide compound employed as a neuropharmacological transport agentmust have --NH₂ groups.

As examples of medicines which may be transported to the central nervoussystem by means of the thiolated polypeptide compound according to theinvention, there may be mentioned: alkaline phosphatase, the A fragmentof cholera toxin, the A fragment of diphtheria toxin, dipyrido-indolesaccording to French patent No. 77,11,148 and generally, any medicinehaving --NH₂ groups.

It is known that the cholera toxin is bound to the GM₁ gangliosides ofthe intestine wall and that the A fragment is responsible for theincrease in the cyclic AMP rate (cyclic adenosine-monophosphoric acid).On the other hand, in tetanus a decrease in the cyclic AMP proportion inthe central nervous system is found. The conjugate according to theinvention, formed by the thiolated polypeptide compound coupled to the Afragment may be employed for controlling tetanus.

The dipyrido-indoles according to French patent No. 77,11,148 arechemotherapeutic agents of utility in the treatment of cancers. In thisfield, it is known that metastases are due to the fact that thecancerous cells come to nestle in the central nervous system hence theymigrate to other regions of the body, where they develop tumors.

The development of metastases could be avoided or reduced provided thatthe means for destroying these cells in the central nervous system canreach the central nervous system.

In the same way, the invention may be applied to the treatment ofcerebral tumors.

The present invention consequently also relates to the means forcoupling the thiolated polypeptide compound according to the inventionto medicines.

The means for coupling the compound according to the invention and themedicine to be transported use at least one disulphide bridge or atleast one sulfur irreversible link.

The present invention therefore also relates to II_(c) fragment medicineconjugates comprising at least one disulphide bridge or at least onesulfur irreversible link.

It is known to prepare protien conjugates by formation of anintermolecular disulphide bridge. The formation of such anintermolecular disulphide bridge is achieved for example by reaction ofa protein having thiol groups with a protein having dithiopyridylgroups.

For example, according to the process described by TE PIAO KING et al[Biochemistry vol. 17 No. 8, 1978], two different proteins may becoupled by first binding thiol groups to one of the proteins and4-dithiopyridyl groups to the other protein and by reacting theresulting modified proteins under suitable conditions in order to form adisulphide bridge and to eliminate 4-thiopyridone. The thiol groups maybe bound to one of the proteins by means of4-methyl-mercapto-butyrimidate and the 4-dithiopyridyl groups to theother protein by means of, for example,3-methyl-(4'-dithiopyridyl)propionimidate. This coupling processproduces a protein-protein conjugate in which the fraction between thetwo proteins is symmetrical with respect to the disulphide bridge.

According to CARLSSON et al. (Bioch. J., 1978, 173, 723-724) the thiolgroup can be introduced in one of the proteins by reaction of saidprotein with N-succinimidyl-3-(2-pyridyl-dithio)propionate andsubsequent reduction; according to this process, the same reagent,namely the N-succinimidyl-3-(2-pyridyl-dithio)propionate, is used forintroducing both thiol and dithiopyridyl groups in the proteins. Theresulting conjugates also have a binding fraction which is symmetricalrelative to the disulphide bridge.

4-methyl-mercapto-butyrimidate has also been used for forming higherdimers and oligomers of proteins of 30 S ribosome of Escherichia Coli(Biochemistry, 12, 3266-3273, 1973).

The conjugates thus obtained have many applications, for example asimmunological assay reagents.

The process according to the invention for coupling the thiolatedpolypeptide compound used as a neuropharmacological transfer agent witha medicine by means of disulphide bridges comprises the steps of

(1)-introducing dithiopyridyl groups in the medicine to be bound;

(2 )-reacting the medicine having the dithiopyridyl groups with thethiolated polypeptide compound according to the invention.

The reaction diagram of this coupling process may be represented in thefollowing manner when the dithiopyridylation agent used in step 1 isN-succinimidyl-3-(2-pyridyl-dithio)propionate: ##STR5##

In this process, another dithiopyridylation agent may be employed, suchas dithiopyridine or any other agent suitable for such a reaction.

Another way of coupling the polypeptide compound, used as aneuropharmacological transport agent according to the invention consistsof creating an irreversible link between said agent and the medicine tobe transported. This process may be represented by the followingreaction diagram: ##STR6##

It comprises the steps of:

(1)reacting the medicine to be bound with the ester ofmethamaleimidobenzoyl-N-hydroxy-succinimide;

(2)reacting the resulting compound with the polypeptide compoundaccording to the invention.

The foregoing reaction diagrams and those which will be givenhereinafter are simplified and do not take into account the number of SHgroups which may be bound to the II_(c) fragment.

It was already proposed to use the ester ofmethamaleimidobenzoyl-N-hydroxy-succinimide for forming enzyme-antibodyconjugates (FEBS Letters, vol.95, No. 2, November 1978). However, theteachings of the prior art did not permit to foresee that the use of theester of metamaleimidobenzoyl-N-hydroxy-succinimide for coupling theneuropharmacological transport agent according to medicines would notmodify or inhibit the pharmacological properties of said medicine andthe property of the II_(c) fragment to be found to the specificreceptors of the tetanus toxin in the central nervous system.

It will be observed that the coupling according to the invention of themedicine to the thiolated polypeptide compound employed as aneuropharmacological transport agent is achieved by known conventionaltechniques of protein-protein coupling. However, it should be noted thatnot all of the protein-protein coupling processes available to oneskilled in the art are suitable for the purposes of the invention.Indeed, only the coupling processes which achieve a disulphide bridge ora sulfur irreversible link are suitable. In particular, it will bementioned that the most conventional coupling process, which employsglutaraldehyde, is unsuitable for the purposes of the invention, sincethe II_(c) fragment treated with glutaraldehyde loses its properties ofaxonal transport and of binding to the receptors of the tetanus toxin inthe central nervous system. Thus, the II_(c) fragment to which carbonylgroups would have been bound, for example with glutaraldehyde, isunsuitable for the purposes of the invention.

Another application of the thiolated polypeptide compound according tothe invention, is the labelling of neuronal cells. Thus, the compound ofthe invention may be employed as a specific labeller of neuronal cells.It is also suitable for preparing immunological reagents. For example anenzymatic reagent may be preparedc from the thiolated polypeptidecompound according to the invention and alkaline phosphatase. It wasfound that the resulting conjugate had both binding power of the II_(c)fragment and the enzymatic activity of the phosphatase. The compoundaccording to the invention is also suitable as a retrogradetranssynaptic tracer.

The polypeptide compound of the invention is also appropriate to preparea diagnosis reagent by coupling with a labelling molecule, such as anantibody, for example a radioactive antibody.

The invention will be now described in more detail by means of theexamples illustrating the preparation of the thiolated polypeptidecompound of the invention and the coupling of the latter with medicines.In all the examples, use was made of the II_(c) fragment as definedhereinbefore.

EXAMPLE 1 Thiolation of the II_(c) fragment by means of 2-iminothiolane

Thiolation of the II_(c) fragment was achieved by means of the methoddescribed by Schramm et al. [Z. Physiol.Chem. 1977, 358; 137-139].

The II_(c) fragment (1.5 mg) in solution in 50% glycerol (0.2 ml) wasthiolated by iminothiolane (2.5 mg) in solution in 750 μl of 0.2 Mtriethanolamine HCl buffer, pH 8.5-9.0. The reaction mixture wasmaintained at room temperature for 2 hours. The excess of reagent waseliminated by filtration on a "SEPHADEX G-25" column. The thiolatedpolypeptide product so obtained contained 2.7-SH groups.

EXAMPLE 2 Thiolation of the II_(c) fragment by means ofN-acetylhomocysteine thiolactone

The II_(c) fragment thiolation was effected by the procedure describedby SINGER et al. [J. Am. Chem. Soc., 1960 82, 567-571[.

To 4 mg of the II_(c) fragment in solution in 0.2 ml of water, there wasadded the K₂ CO₃ /NaHCO₃ buffer, pH 10.7 (0.2 ml). A nitrogen currentwas passed therethrough in order to expel the air. 0.2 ml of an 80 mg/lsolution of N-acetylhomocysteine thiolactone was added. The reactionmixture was maintained for two hours at 4° C. under nitrogen. Thereaction was thereafter stoppd by filtration on SEPHADEX G-25. The thusobtained product contained 4-SH groups.

EXAMPLE 3 Preparation of a conjugate of thiolated polypeptide compound/A fragment of the diphteria toxin

5 mg of the A fragment of diphteria toxin were used in 1.5 ml of 0.1 Mphosphate buffer containing 0.5 M NaCl (pH 7.5). There was added 312 μgof N-succinimidyl-3-(2-pyridyldithio)propionate (Pharmacia) dissolved in25 μl of 99.5% ethanol (Merck). After reacting for 30 minutes in a waterbath at 23° C., the dithiopyridylated toxin was separated from theexcess of reagent by filtration on "Sephadex G 25" buffered with thesame buffer.

This procedure achieved introduction of 1.7 2-pyridyldisulphide groupingper mole of A fragment.

The II_(c) fragment was thiolated by reaction with4-methyl-mercaptobutyrimidate according to the method described by TEPIAO KING et al. (Biochemistry, vol. 17, No. 8, 1978), 5.37 mg of II_(c)dissolved in 1.5 ml of 0.025 M borate buffer, pH 9.0, were reacted with3 mg of the thiolation agent dissolved in 100 μl of methanol at 0° C.for 30 minutes. The excess of reagent was removed by filtration on"Sephadex G 25" balanced with a 0.1 M phosphate buffer, pH 7.0,containing 1 mM Na₂ EDTA. The thiolated II_(c) fragment contained 2.5-SHgroups per mole.

The conjugate was obtained by mixing 119 nanomoles of dithiopyridylatedA fragment with 90.9 nanomoles of thiolated II_(c) fragment. Theexchange reaction was followed at 343 nm. Filtration was effected on"Sepharose 6B" buffered with a buffer Tris 0.05 M, 0.5 M NaCl, pH 8.0.

The conjugate thus obtained preserves the binding power of the II_(c)fragment and the immunological reactivity of the A fragment and of theII_(c) fragment.

FIGS. 1A and 1B illustrate the synthesis of the II_(c) -polypeptideconjugate. They represent the result of two gel immunodiffusionexperiments.

The photograph in the left part of FIG. 1A shows the specificimmunological reaction between the TT (tetanus toxin)fragment and theantitetanus serum disposed in the central well. No reaction occurs withthe A fragment of the diphteria toxin. The A fragment of the diphteriatoxin, which has an antigenic structure different from that of thetetanus toxin, exhibits no precipitation line when contacted withantitetanus serum.

In FIG. 1A, the [A-SS-II_(c-) ] compound, viz. the thiolated II_(c)previously coupled with the A fragment of diphterial toxin, is reactingwith the antitetanus serum (ATS) through its tetanus antigenicdeterminants.

In FIG. 1B, the [A-SS-II_(c-) ] compound is reacting with antidiphtheriaserum (ADS) through the antigenic determinants borne by the A fragmentof the diphterial toxin.

Thus, the photographs in FIGS. 1A and 1B show that synthesis ofA-SS-II_(c) conjugate occurred and that the two components, viz. the Afragment and the II_(c) fragment, retained their antigenic propertiesafter the coupling.

EXAMPLE 4 Pharmacological experiments

In these experiments, the II_(c) fragment, the thiolated polypeptidecompound according to the invention, as well as the tetanic toxin andthe other fragments used for comparison purposes, such as fragmentB-II_(b), were labelled with 125 iodine. This labelling with 125 iodinewas made according to the method of GREENWOOD et al. [The preparation of¹²⁵ I-labelled human growth hormone of high specific radioactivity.Biochem. J. 89, (1963) 114-127].

The specific radioactivity of the ¹²⁵ I-labelled II_(c) fragment used inthe retrograde transport experiments was of 4 μCi per μg of protein. TheII_(c) fragment used for testing the binding to synaptic membranes had aspecific radioactivity of 1.2 μCi per μg of protein. The specificradioactivity of the tetanic toxin was of 2.5 μCi per μg of protein.

(A)II_(c) fragment binding to crude synaptic membranes

The crude synaptic membranes were prepared from the spinal cord of maleSprague Dawley rats of a weight of 150-200 g according to the Young andSnyder method, Strychnine binding associated with glycine receptors ofthe central nervous system. Proc. Nat. Acad. Sci. U.S.A. 70, (1973)2832-2836. The protein concentration in the membrane suspension wasadjusted to 1.0 mg per ml. The suspension was stored in aliquots of 1 mlat -25° C. The tests were effected according to the above-mentionedYoung and Snyder method. The II_(c), B-II_(b) fragments, or the toxin,were caused to react with aliquots of 0.1 mg of crude synaptic membranesfor 15 minutes at 21° C. in 1 ml of 0.05 M Na/K phosphate buffer, pH7.4, containing 0.01% of "Triton X-100" The reaction was terminated bycentrifuging during 10 minutes at 48,000×g and 4° C. The supernatentfluid was decanted and the sediment was washed three times with 5 ml of0.05 M Na/K phosphate buffer, pH 7.4, containing 0.01% of "Triton X-100"and 0.1% of bovine serum albumin. The radioactivity of the supernatent,of the washing liquors and of the sediment was then measured. Each testwas repeated three times. The tests were also effected with unlabelledII_(c) or B-II_(b) fragments or with unlabelled toxin. In this case, afixed amount of labelled protein was incubed with increasing amounts ofunlabelled proteins.

It was ascertained that binding of the ¹²⁵ I-labelled II_(c) fragmentwas increasing linearly with the membrane concentration.

Displacement of the ¹²⁵ I-II_(c) fragment bound to the isolated synapticmembranes by the unlabelled II_(c) fragment or labelled tetanus toxinand displacement of the non ¹²⁵ I-labelled tetanus toxin by theunlabelled II_(c) or B-II_(b) fragments were measured. The resultsobtained are shown in FIGS. 2A and 2B wherein there was plotted asordinates the specific binding of the ¹²⁵ I-II_(c) fragment (FIG. 2A) asexpressed in cpm (counts per minute) or of the ¹²⁵ I-labelled tetanustoxin (FIG. 2B) and as abscissae the molar concentration (M) of II_(c),B-II_(b) fragments or of tetanic toxin.

The half maximal displacements were computed from the curves in FIGS. 2Aand 2B and are gathered in table I.

The results in table I show that the half maximal displacement of the¹²⁵ I-II_(c) fragment bound to synaptic membranes has approximately thesame value whether it is caused by the unlabelled II_(c) fragment or bythe whole tetanus toxin. The results in table I also show that theunlabelled II_(c) fragment is as much effective to displace ¹²⁵I-labelled tetanus toxin as the unlabelled toxin is to displace the ¹²⁵I-II_(c) fragment.

In contrary, the B-II_(b) fragment is twice more effective than theII_(c) fragment to displace the 125 iodine-labelled tetanus toxin boundto synaptic membranes.

The Hill transformation of the displacement of the ¹²⁵ I-II_(c) fragmentbound to isolated synaptic membranes by the unlabelled II_(c) fragment,expressed as ##EQU1## of the concentration of the unlabelled II_(c)fragment provides a linear curve (FIG. 3) with a slope of 1.68 which maybe construed as showing a positive cooperativity according toCornish-Bowden et al., Diagnostic uses of the Hill (Logit and Nernst)plots. J. Mol. Biol. 95 (1975) 201-212.

The same tests as above were effected with the thiolated II_(c) fragmentaccording to the invention obtained according to the above Example I.The results obtained, also shown in table I, show that thiolation of theII_(c) fragment according to the invention does not substantially modifythe properties of fragment II_(c) to be bound to synaptic membranes.

(B) Axonal retrograde transport Experimental procedure

For all experiments, female Sprague-Dawley rats weighing 250 g wereused. The rats were maintained at a constant temperature of 23° C. andfed with the usual diet (Nafage Gossau) and water.

Two albino rats received an injection of the ¹²⁵ I-II_(c) fragment andof 30% horse-radish peroxydase (HRP)

As references, two other rats received an injection of the B-II_(b)fragment (1 μl corresponding to a concentration of 0.7 μg of protein perμl).

After baring the muscle, the substances were injected by an thermallycontrolled injection system by means of a glass pipette (50-100 μm inoutside diameter). The injection time was about 40 minutes.

The total injected volume was 2.5 μl, viz. 1.5 μl of II_(c) fragmentsolution with 3 μg of protein per μl, and 1 μl of HRP solution.

The rats were sacrificed 24 hours after the injection. An intracardialperfusion was effected under general anesthesia first with 0.5 ml of"Liquemine" (Roche) and 0.5 ml of sodium nitrite (0.01 g/ml), then with200 ml of a plasma expander ("MACRODEX") for 5 minutes, thereafter with600 ml of 1% paraformaldehyde and 2.5% glutaraldehyde in a 0.1 Mphosphate buffer for 30 minutes, and lastly with a 10% sucrose solutionin a 0.1 M phosphate buffer for 20 minutes.

For more details about this procedure, reference may be made to thearticle of MESULAM [J. Histochem. Cytochem., 26 (1978) 106-117].

The brain was removed immediately after this perfusion and placed in a30% sucrose solution for 48 hours before being cut. Frozen sections (30μm in thickness) were taken, starting from the caudal end of theabducens nucleus up to the rostral end of the whole oculomotor nucleus.Each section treated with the horse-radish peroxidase was stained by theTMB method of MESULAM and restained with neutral red while the othersections were autoradiographed. The latter sections were mounted anddipped into a liquid emulsion NTB₂ at 45° C. diluted 1:2 with distilledwater. The sections were exposed for 4 weeks at 4° C. in the dark anddeveloped with "Kodak Dektol" developer at 18° C. for 90 seconds,washed, and then fixed with 30% sodium thiosulfate, washed for 2 hours,then stained with cresyl violet and covered. All the sections were thenexamined under the microscope (250 magnification) and the location ofthe labelled cells was ascertained by microphotography.

As the B-II_(b) fragment, the II_(c) fragment undergoes axonalretrograde transport since positive labelling occurs in the oculomotornucleus of the two tested animals after the injection of HRP and of theII_(c) fragment, as evidenced bu photographs of the oculomotor complexwith a radioactive labeller taken in the region under consideration.

The main difference between the HRP and the II_(c) fragment resides inthe fact that the location of the HRP granules is limited to theperikarya and dendrites of the oculomotor neurones, while the silvergrains which represent the ¹²⁵ I-II_(c) fragment were also found in thepericellular spaces.

As compared with the B-II_(b) fragment and conjugates thereof, theII_(c) fragment seems to be more strictly located in the cellular bodiesof the labelled motor neurones than in the extra cellular spaces. Whilea higher total activity was injected in both cases, with the II_(c)there was noted a less intensive labelling of the oculomotor nucleusthan with the B-II_(b) fragment.

The fact that the axonal retrograde transport is in direct relationshipwith the ability of the fragments to be bound to their membranereceptors clearly shows that the thiolated II_(c) fragment which haskept his binding properties (Table I) also undergoes retrogradetransport.

By means of the tests described in the Journal of Neurochemistry (1977)vol. 28, p. 529-542, it was verified that the II_(c) medicine conjugatesobtained according to the invention from the thiolated polypeptidecompound were bound in a specific manner to the gangliosides and thesynaptic membranes; these results demonstrate that these substances canconsequently be transported in retrograde axonal manner.

                  TABLE I                                                         ______________________________________                                        half maximal displacements per mg of proteins of membrane                     as computed from the curves in FIGS. 1A and 1B                                Displacement    half-maximal displacement nM/mg                               ______________________________________                                        .sup.125 I-II.sub.c -                                                                         8.0                                                           unlabelled II.sub.c fragment                                                  .sup.125 I-II.sub.c -                                                                         9.0                                                           unlabelled tetanic toxin                                                      .sup.125 I - tetanic toxin-                                                                   9.0                                                           unlabelled II.sub.c fragment                                                  .sup.125 I - tetanic toxin-                                                                   5.5                                                           unlabelled B-II.sub.b fragment                                                .sup.125 I- thiolated I-II.sub.c                                                              9.5                                                           unlabelled II.sub.c fragment                                                  ______________________________________                                    

What I claim is:
 1. A thiolated polypeptide compound constituted by the II_(c) fragment of tetanus toxin to which at least one --SH group is directly or indirectly bound through the amino groups thereof, said compound being obtained by thiolation and comprising at least one Z-SH group, wherein Z is the residue of a thiolation agent and is chosen from the group consisting of: ##STR7##
 2. II_(c) fragment of tetanus toxin/medicine conjugate comprising at least one disulfide bridge, said medicine having amino groups.
 3. II_(c) fragment of tetanus toxin/medicine conjugate comprising at least one irreversible --S-- link, said medicine having amino groups.
 4. II_(c) fragment/medicine conjugate according to claim 2 wherein the medicine is the A fragment of cholera toxin, the A fragment of diphtheria toxin, or a dipyrido-indole.
 5. The thiolated polypeptide compound claimed in claim 1, wherein the number of said Z--SH groups is not greater than about
 5. 6. Method of administration of the thiolated polypeptide compound according to claim 1 as an axonal retrograde transport agent for transporting a medicine having amino groups to the central nervous system of a mammal. 